PSG Center for Molecular Medicine & Therapeutics, PSG Institute of Medical Sciences & Research, Coimbatore, 641 004, Tamil Nadu, India.
Marine Biotechnology Division, National Institute of Ocean Technology, Chennai, 600100, Tamil Nadu, India.
Biotechnol Lett. 2020 Aug;42(8):1527-1534. doi: 10.1007/s10529-020-02873-6. Epub 2020 Apr 3.
Ubiquitination has a role as a host defense mechanism against pathogens. To channelize autophagic mycobacteria to destruction, ubiquitin ligase like, Makorin Ring Finger Protein 1 (MKRN1) was speculated to play a role in ubiquitinating M. tuberculosis. We have developed a flow cytometry based in vitro ubiquitin ligase assay to understand the role of MKRN1 in ubiquitinating mycobacteria and confirmed the results by western blotting.
MKRN1 was cloned and expressed in E. coli BL21 (DE3) strain. The recombinant MKRN1 protein was solubilised, purified and refolded to restore the activity. In addition, through autoubiquitination assay, the activity of protein was confirmed. The corresponding E1 and E2 enzymes for MKRN1, UBE1 and UBE2D3 respectively, were selected using BioGrid tool. Surprisingly, flow cytometric assay revealed that at a concentration of 300 nM of MKRN1, 38% of M. tuberculosis was found to be ubiquitinated in vitro with 3.5% of the cells having bound MKRN1. Immunoblot results also substantiates the ubiquitination of M. tuberculosis. MKRN1 did not ubiquitinate B. Subtilis and therefore, we speculate that the E3 Ub ligase activity might be specific to M. tuberculosis.
This clearly demonstrates that recombinant MKRN1 ubiquitinates M. tuberculosis which opens up a novel, potential role of MKRN1 against mycobacteria which has to be unfolded.
泛素化在宿主防御机制中具有对抗病原体的作用。为了将自噬分枝杆菌导向破坏，推测泛素连接酶样 Makorin 环指蛋白 1 (MKRN1) 在泛素化结核分枝杆菌中发挥作用。我们开发了一种基于流式细胞术的体外泛素连接酶测定法，以了解 MKRN1 在泛素化分枝杆菌中的作用，并通过 Western blot 验证了结果。
克隆并在大肠杆菌 BL21 (DE3) 菌株中表达了 MKRN1。重组 MKRN1 蛋白可溶解、纯化和重折叠以恢复其活性。此外，通过自动泛素化测定法，确认了蛋白质的活性。分别使用 BioGrid 工具选择了用于 MKRN1 的相应 E1 和 E2 酶，即 UBE1 和 UBE2D3。令人惊讶的是，流式细胞术检测显示，在 300 nM 的 MKRN1 浓度下，体外有 38%的结核分枝杆菌被泛素化，有 3.5%的细胞与 MKRN1 结合。免疫印迹结果也证实了结核分枝杆菌的泛素化。MKRN1 未泛素化枯草芽孢杆菌，因此我们推测 E3 Ub 连接酶活性可能是分枝杆菌特有的。
这清楚地表明重组 MKRN1 泛素化结核分枝杆菌，这为 MKRN1 针对分枝杆菌开辟了一个新的、潜在的作用，这一作用还有待进一步阐明。
